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Late blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes.
We previously cloned a late blight resistance gene, RB, from a diploid wild potato species Solanum bulbocastanum. Transgenic potato lines containing a single RB gene showed a rate-limiting resistance against all known races of Phytophthora infestans, the late blight pathogen. To better understand the RB-mediated resistance we silenced the potato Rar1 and Sgt1 genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The Rar1 and Sgt1 genes of a RB-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the Rar1 and Sgt1 silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged P. infestans strain. The resistance of the Rar1-silenced plants was not affected. However, silencing of the Sgt1 gene abolished the RB-mediated resistance.
Our study shows that silencing of the Sgt1 gene in potato does not result in lethality. However, the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, the Rar1 gene is not required for RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.
Potato late blight, a disease caused by the oomycete pathogen Phytophthora infestans, is one of the world's most devastating crop diseases. World-wide losses due to late blight exceed several billion dollars annually [1]. Most of the potato cultivars currently grown in the United States are highly susceptible to late blight and control of this disease relies almost exclusively on fungicide applications. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. The pedigrees of many potato cultivars currently used in different countries include late blight resistant germplasm derived from Solanum demissum, Solanum andigena, and other wild species. However, most of the resistance derived from these wild species is controlled by single dominant resistance genes (R genes). These R genes are only effective in preventing the development of late blight if the invading P. infestans race contains the corresponding avirulence genes. This R gene-mediated resistance is often short-lived and is rapidly overcome by new races of the late blight pathogen.
Solanum bulbocastanum (2n = 2x = 24) is a diploid species that has adapted in the same environment as the late blight pathogen. This wild species was characterized as possessing durable resistance against P. infestans, even under high disease pressure [2,3]. Two resistance genes, RB (Rpi-blb1) and Rpi-blb2, have been cloned from S. bulbocastanum [4-6]. Both genes confer broad-spectrum resistance against a wide range of known P. infestans races. Transgenic potato lines containing a single RB gene showed a high-level resistance in the Toluca Valley, Mexico, where the potato fields are naturally intensively infested with the most diversified P. infestans populations [7]. Most interestingly, transgenic RB plants did not show total immunity to late blight, but instead showed a marked delay in both onset of symptoms and development of lesions. Such rate-limiting resistance may put less selection pressure on the P. infestans populations and protect the durability of this resistance gene. The RB gene therefore provides an excellent model to study the mechanism of broad-spectrum and rate-limiting disease resistances. An understanding of the underlying mechanism of this type of resistance is important for developing strategies to breed durable and sustainable disease resistance.
Several genes have been implicated in the regulation of R gene function. Of these genes, Rar1 and Sgt1 are among the most extensively studied genes. The Rar1 (required for Mla12 resistance) gene was first identified for its essential role in the function of a subset of Mla genes that confer resistance to barley powdery mildew [8]. The RAR1 protein contains two highly similar but distinct cysteine- and histidine-rich (CHORD) Zn2+-binding domains and was proposed to play a role in stabilizing R proteins in a confirmation that is implicated in receiving pathogen signals [9]. The Sgt1 gene (suppressor of the G2 allele of skp1) is an essential gene with multiple functions in yeast. SGT1 protein was initially identified as a RAR1-interacting partner in a yeast two-hybrid screen [10]. SGT1 may play a role in R protein accumulation [11]. Rar1 and Sgt1 genes are required in various R-gene mediated resistance against viral, bacterial, oomycete or fungal pathogens [12]. However, none of the previously studied R genes showed a race-non-specific and rate-limiting resistance phenotype as the RB gene. In addition, the role the Rar1 and Sgt1 genes are not universal and these genes are not essential for resistance involving some R genes [12,13].
Besides the two broad-spectrum resistance genes RB and Rpi-blb2, several race-specific late blight resistance genes have also been cloned [14-16]. Numerous late blight resistance genes have recently been mapped in various potato species or populations [17-25]. However, there is almost no information available about the resistance pathways mediated by any of these genes. As an initial effort to understand the RB-mediated late blight resistance pathway, we silenced the Rar1 and Sgt1 genes using an RNAi-based approach in a potato line containing the RB gene. We demonstrated that SGT1, but not RAR1, is essential for the RB-mediated broad-spectrum resistance to potato late blight.
Identification of the potato Rar1 and Sgt1 genes
A search of the Institute for Genomic Research (TIGR) potato database [26] using a sequence from the tobacco Rar1 gene (AF480487) identified a potato EST, TC121848 (1187 bp), which showed 96% sequence similarity to the tobacco and tomato Rar1 transcripts. Similarly, a search using a sequence from the tomato Sgt1 gene (TC85297) identified a potato EST, TC112395 (1461 bp), which showed 98% sequence similarity to the tobacco and tomato Sgt1 genes and 90% sequence similarity to the Arabidopsis thaliana Sgt1b gene. Since the full-length cDNAs for the A. thaliana Rar1 and Sgt1b genes are 901 and 1290 bp, respectively [27,28], the identified potato ESTs almost cover each of the complete potato genes. Southern blot hybridization was performed to determine the copy numbers of the Rar1 and Sgt1 genes in the potato genome. Genomic DNA was isolated from potato clone K41, which contains the RB gene introgressed from S. bulbocastanum, and was hybridized with the potato Rar1 and Sgt1 gene probes. The Southern hybridization results showed that the haploid potato genome contains only one copy of the Rar1 gene and two copies of the Sgt1 gene (data not shown), which agree with a similar conclusion reported by Pajerowska et al. (2005).
RNAi-based silencing of the potato Rar1 and Sgt1 genes
We developed RNAi constructs for the potato Rar1 and Sgt1 genes. The Rar1 silencing construct contained a 474-bp fragment covering the CHORD II domain. The Sgt1 construct contained a 481-bp fragment that targets the P23/CS domain (Figure 1). These constructs were used for Agrobacterium-mediated transformation of the potato line K41. We obtained 65 and 58 independent transgenic lines for the Rar1 and Sgt1 genes, respectively.
Figure 1. Schematic representation of the potato Rar1 and Sgt1 genes and regions used for RNAi construct development.
The expression of the Rar1 and Sgt1 genes in the transgenic lines was analyzed by Northern blot hybridization using the Rar1 (1187 bp) and Sgt1 (1461 bp) genes as probes. A significant reduction of the Rar1 transcripts was observed in 47 of the 65 Rar1-RNAi transgenic lines analyzed (Table 1). Sgt1 transcript reduction was also observed in 35 of the 58 Sgt1-RNAi lines (Table 1). Three Rar1-RNAi lines and one Sgt1-RNAi line showed no detectable Northern hybridization signals after exposure for over one month using intensifying screens (Figure 2). Only these four lines were used in the late blight resistance evaluation. We observed no distinguishable morphological features associated with RNAi-induced silencing of the Rar1 and Sgt1 genes in these transgenic lines.
Figure 2. Transcription analysis of Rar1 and Sgt1 silenced transgenic lines. (A) Northern blot analysis of transcription of the Rar1 gene. Lane 1: a transgenic plant (clone 3007) containing the Rar1-RNAi construct but not silenced; Lane 2–4: three independent Rar1-silenced lines (clones 3128, 2998, 3028) showing no transcript; Lane 5: untransformed K41 control. (B) Northern blot analysis of transcription of the Sgt1 gene. Lane 1: a transgenic plant (clone 3061) containing the Sgt1-RNAi construct but not silenced; Lane 2: Sgt1-silenced line (clone 3095) showing no transcript; Lane 3: untransformed K41 control. Both blots were re-probed with an actin gene probe to ensure equal loading of mRNA in each lane.
Table 1. RNAi Silencing efficiencies of the Rar1 and Sgt1 genes based on Northern blot hybridization
Late blight resistance evaluation of the Rar1- and Sgt1-silenced potato lines
We evaluated the late blight resistance of the Rar1- and Sgt1-silenced lines together with several controls that either contain or do not contain the RB gene, including transformed but not silenced Rar1-RNAi and Sgt1-RNAi lines (no transcript reduction was found in these RNAi lines). Triplicates of each line were used in each of two independent inoculation experiments. All variation within replicates and experiments were taken into account in determining resistance scores. The average score of the late blight infection on the Rar1-silenced plants was 7.3 (± 0.6) after 7 days post inoculation (dpi) and 6.9 (± 0.0) after 10 dpi, which represents ~13% and 18% foliage infection, respectively (Figures 3, 4). The untransformed K41 plants and the non-silenced Rar1-RNAi line showed an average score of 8.0 (± 0.0), representing less than 10% infection. All susceptible controls showed an average score of 2.1 (± 0.6), representing ~81% infection. ANOVA showed that there was a significant difference in mean resistance scores among the tested plants (P-val = 2.2e-16) after seven days. Fisher's Least Significant Difference (LSD) test as a comparison of resistance score means revealed no significant differences between the Rar1-silenced plants and other resistant controls but revealed significant differences with the susceptible control, Katahdin. These results show that the RB-mediated resistance was not affected in the Rar1 silenced lines.
Figure 3. Late blight resistance phenotype of Rar1- and Sgt1-silenced lines. Controls and silenced plants were inoculated with P. infestans and photographs were taken seven days after inoculation. (A, F) Untransformed K41 control (RB+); (B) A transformed but not silenced Rar1-RNAi line (clone 3007); (C, D) Two independent Rar1-silenced lines (clones 3128, 2998); (E, I) Susceptible control Katahdin (RB-); (G) A transformed but not silenced Sgt1-RNAi line (clone 3061); (H) A Sgt1-silenced line (clone 3095).
Figure 4. Late blight resistance evaluation of Rar1- and Sgt1-silenced lines. Resistance readings were taken 5, 7 and 10 days post inoculation (dpi). K41: untransformed control (RB+); Katahdin: susceptible control (RB-); PT29: a S. bulbocastanum clone as the resistant control (RB+); Non-silenced Rar1 line (RB+): a transgenic, but non-silenced Rar1-RNAi line (clone 3007); Rar1 RNAi line #1 (Rar1 silenced, RB+, clone 3128); Rar1 RNAi line #2 (Rar1 silenced, RB+, clone 2998); Sgt1 RNAi line (Sgt1 silenced, RB+, clone 3095); Non-silenced Sgt1 line (RB+): a non-silenced Sgt1-RNAi line (clone 3061). Error bars represent the standard deviation from the means.
The Sgt1-silenced plants showed an average late blight infection of 4.0 (± 0.0) after 7 dpi and 3.7 (± 0.7) after 10 dpi, representing ~70% and ~73% infection, respectively (Figures 3, 4). Significant differences among the mean resistance scores were observed among the tested plants starting from day seven using ANOVA with P-val = 2.2e-16. Fisher's LSD test revealed that the Sgt1-silenced plant had a significantly different resistance score compared to the resistant control (untransformed K41) and non-silenced Sgt1 plants. These results suggested that silencing of the Sgt1 gene compromised the RB-mediated late blight resistance.
The late blight resistance of the Rar1- and Sgt1-silenced lines were also evaluated using the GFP-tagged P. infestans isolate 208m2 [29]. The Phytophthora growth was quantified by counting the fluorescing sporangia within each area; more P. infestans growth is indicated by larger counts. One and three days after inoculation, no significant differences were observed between the sporangial counts on the silenced and non-silenced controls. Six days after inoculation, however, there were significant differences among the sporangial counts of the tested plants (Figure 5) based on a one-way ANOVA with unequal variance (F = 3.627, P-val = 0.03142). Fisher's LSD, using an alpha of 0.01, indicated that the Sgt1-silenced plant had significantly more P. infestans growth, as indicated by sporangial count, than the K41 control, the transgenic but not silenced Rar1-RNAi and Sgt1-RNAi lines and the Rar1-silenced plants (Figure 6). These results are consistent with those from the conventional greenhouse evaluations.
Figure 5. Late blight resistance evaluation using a GFP-tagged P. infestans strain. Leaves were inoculated on plants with GFP-tagged isolate 208m2 and were removed 6 days post inoculation and photographed on a dissecting microscope under a narrow-band GFP filter. (A) Untransformed K41 control. (B) Katahdin. (C) A silenced Rar1-RNAi line (clone 3128). (D) A Sgt1-silenced line (clone 3095). The red circles outline the original location of the 10 μl inoculation drops. Green fluorescence is only observed within the circles in A and C but spreads out of the original inoculation sites in B and D. Bars = 2.5 mm.
Figure 6. Quantitative analysis of GFP Phytophthora lesions. The average GFP sporangial counts (particle count) are shown from K41: untransformed control (RB+); Non-silenced Rar1 line (RB+): a transgenic, but non-silenced Rar1-RNAi line (clone 3007); A Rar1-RNAi line (clone 3128); Non-silenced Sgt1 line (RB+): a non-silenced Sgt1-RNAi line (clone 3061); A Sgt1-RNAi line (clone 3095). Using a one-way ANOVA, with unequal variance, and Fisher's LSD at an alpha of 0.01, the only group that has significantly higher sporangial growth is the Sgt1-silenced line.
The Rar1 and Sgt1 genes in potato
Rar1 is a single copy gene in barley [10] and A. thaliana [28,30]. Our data from Southern blot hybridizations confirm the previous report that only one copy of Rar1 exists in the haploid potato genome [31]. Plant rar1 mutants show no visible growth defects [32], although silencing of the Rar1 homolog in C. elegans resulted in semi sterility and embryo lethality [8]. The Rar1-silenced potato plants did not show any visible growth phenotypes in our study.
SGT1 is an essential protein for proper kinetochore function in yeast and null mutations of the single copy Sgt1 gene are lethal [33]. Most plant species analyzed appear to contain two Sgt1 genes [11,31,34,35]. The Sgt1a and Sgt1b double mutant in A. thaliana is embryo lethal [11]. Similarly, virus-induced gene silencing (VIGS) of the Sgt1 genes (NbSgt1.1 and NbSgt1.2) in N. benthamiana caused a stunt phenotype [34]. In tomato, silencing of the Sgt1-1 gene, but not Sgt1-2, was lethal. This result was possibly caused by silencing of both the Sgt1-1 and Sgt1-2 genes using the VIGS construct designed for Sgt1-1 [35]. In contrast, silencing of the wheat and barley SGT1 genes by VIGS did not result in a growth phenotype [36,37]. However, the copy number and functional redundancy of the SGT1 genes in wheat and barley are not clear, this may be caused by either partial silencing of the gene and/or compensated by partially homologous and non-silenced SGT genes in these species.
We did not observe any abnormal growth phenotype in the Sgt1 silenced line. The complete silencing of the Sgt1 gene was confirmed by Northern blot hybridization (Figure 2B). The available sequences of the two potato Sgt1 genes (StSgt1-1: AY615272; StSgt1-2: AY615274) are 100% identical. The Sgt1 cDNA fragment used in our RNAi contruct also shares 100% homology to these sequences. These results suggest that both copies of the potato Sgt1 gene are likely silenced in the RNAi silencing line. Therefore, it appears that silencing both of the Sgt1 genes is not lethal in potato. This result will need to be confirmed by multiple completely silenced Sgt1 lines.
Requirement of the RAR1 and SGT1 proteins in disease resistance
A role of the RAR1 protein in disease resistance signaling was first recognized in barley through Mla12-mediated resistance against powdery mildew [8]. The same role for RAR1 has since been identified in the resistance pathways conferred by several genes that belong to the NB-LRR family [12,36-38]. Interestingly, there were also reports that the Rar1 gene appears to not play a role in the resistance pathway. The RAR1-independent cases include the Mla1-mediated resistance against powdery mildew in barley [10,39], Bs2/AvrBs2-mediated resistance against bacterial spot disease in N. benthamiana [40], and Mi-1-mediated resistance against root-knot nematodes in tomato [35]. We demonstrated that silencing of the single Rar1 gene in potato does not affect the RB-mediated late blight resistance.
The previous RAR1-independent conclusions were based on gene silencing techniques by biolistic delivery of double stranded RNA constructs into single cells [10,39] or by VIGS [35,40]. These techniques may not result in complete suppression of a target gene and uniform silencing of the gene throughout the infected plants. False negative results have to be carefully sorted out because a low level of the transcripts resulting from incomplete silencing may be sufficient to facilitate the function of RAR1. The RNAi-silenced Rar1 potato lines showed no transcripts based on stringent Northern blot hybridizations (Figure 2), suggesting a near complete silencing of the Rar1 gene. These stable silenced lines allowed repeated resistance evaluations. Thus, our results from multiple RNAi silencing lines provide conclusive evidence that RB-mediated resistance is RAR1 independent.
The RAR1 protein has been proposed to be involved in forming or stabilizing the recognition complexes associated with R proteins or in assisting conformational changes of the recognition complexes [12]. Such functions may not be required in the complex assembly of a subset of R proteins. Bieri et al (2004) showed that in barley rar1 mutants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. However, MLA6 accumulated to about a four-fold lower level than MLA1 in transgenic lines. Interestingly, Mla1 functions independently of Rar1 only where MLA1 abundance exceeds a threshold level [39]. A number of previous studies suggest that RAR1 may control the abundance of the NB-LRR type R proteins [39]. We propose that the RB protein level in the Rar1 silenced potato lines may be sufficient to trigger the resistance pathway.
Many previous studies confirmed the requirement of the SGT1 protein in resistance mediated by both NB-LRR and Pto-kinase type resistance genes, as well as in non-host resistance [34,35,37,40,41]. Several resistance genes in A. thaliana were SGT1b independent in the sgt1b mutant background [27,28,30]. However, SGT1a may complement the loss of SGT1b [11]. The MLA1-triggered resistance in barley, unlike other MLA variants, was largely unaffected when the HvSgt1 gene was silenced [10]. The transient silencing method used in this system may not have been complete, leaving some levels of HvSGT1 for MLA1 to function. Alternatively, MLA1 requires low levels of HvSGT1 to operate as compared to other MLA variants [10]. Bhattarai et al. (2007) recently showed that partial silencing of the Sgt1-1 gene in tomato resulted in attenuation of Mi-1-mediated potato aphid resistance, but the same plants still held the Mi-1-mediated root-knot nematode resistance. These results support the hypothesis that plant R proteins differ in the amounts of SGT1 needed to trigger effective resistance [11]. We demonstrate that silencing of Sgt1 clearly compromised the RB-mediated late blight resistance (Figures 3, 4, 5), indicating that the RNAi approach most likely silenced both copies of the Sgt1 gene and the Sgt1 gene plays the key role in the pathway. Our results provide further evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.
We developed RNAi-based silencing lines of genes Rar1 and Sgt1 in a potato line that contains the late blight resistance gene RB, which confers broad-spectrum resistance against all known races of P. infestans. Silencing of the Rar1 and Sgt1 genes in potato does not result in phenotypic changes. Late blight resistance evaluation of the Rar1 and Sgt1 silenced lines showed that the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, silencing of the Rar1 gene does not abolish the RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.
Plant materials
Potato line J101K6A6K41 (K41) is a tetraploid clone and was developed from the somatic hybrid J101 between potato and S. bulbocastanum (clone PT29) [42]. J101 was used as the female parent to backcross with Katahdin (BC1), Atlantic (BC2) and Katahdin (BC3). K41 is a late blight resistant BC3 clone. Presence of the RB gene in K41 was confirmed by polymerase chain reaction (PCR) using RB-specific primers [43] and this clonal line was used in transformation experiments designed to silence the Rar1 and Sgt1 genes. S. bulbocastanum (PT29) and potato cultivar 'Katahdin' were used as controls for late blight resistance evaluation.
RNAi construct design and potato transformation
Total RNA was extracted from leaf tissue of K41 using the RNeasy Plant Mini Kit (Qiagen, Valencia, California) and treated with TURBO DNA-free (Ambion, Austin, Texas) to remove DNA contamination. First strand cDNA was synthesized using 1 μg of total RNA, oligo d(T) primer and superscript reverse transcriptase (Invitrogen, Carlsbad, California). The cDNA fragments used to silence Rar1 and Sgt1 were amplified by PCR. A 474-bp cDNA fragment from Rar1, corresponding to the TIGR potato EST TC121848 (nt 346 through 820), was amplified from K41 cDNA using Platinum Taq DNA polymerase (Invitrogen). The primers used for amplification included 5' CACC CAA CAC CAT CTG CTA CCA AAA A 3'(forward) and 5' GAC ACT GGG TCA GCG TTG TG 3'(reverse). A 481-bp cDNA fragment from Sgt1, corresponding to the TIGR potato EST TC112395 (nt 359 through 840 bp) (this EST is recently split into TC133190 and TC159283) was amplified from K41 cDNA using primers 5' CACC GGC CTG TAT GAA GCT TGA AGA A 3'(forward) and 5' TCT GCA TTT TGC AGG TGT TAT C 3' (reverse). The Rar1 and Sgt1 amplicons were successively purified using QIAquick PCR purification kit (Qiagen), gel verified and then cloned into pENTR/D-TOPO vector using the pENTR Directional TOPO Cloning kit (Invitrogen). The Rar1 and Sgt1 DNA fragments were then transferred into the pHellsGate8 vector using the LR Clonase recombination method according to Helliwell et al. (2002) [44]. The sequence-verified pHellsgate8-Rar1 and pHellsgate8-Sgt1 constructs were then transformed into K41 using standard Agrobacterium-mediated transformation protocols [45].
Gel-blot hybridizations
Southern blot hybridization was performed according to Stupar et al. (2002) [46]. Genomic DNA was isolated from leaf tissues of K41 and digested with restriction enzymes. The DNA blots were probed with a 1-kb genomic DNA fragment of the Rar1 gene and a 1.1-kb genomic DNA fragment of the Sgt1 gene to evaluate the copy numbers of these two genes in the potato genome. RNA blots were prepared using 10–15 μg of total RNA isolated from leaf tissue using the TRIzol protocol (Invitrogen). To verify the transcription of the Rar1 and Sgt1 genes in the RNAi lines, RNA blots were hybridized with the complete cDNA fragments of Rar1 (TC121848) and Sgt1 (TC112395) amplified from K41 cDNAs. Hybridizations were performed as described previously [47]. Intensifying screens were used to detect the Northern blot hybridization signals and the blots were exposed for at least one month to reveal if any transcripts were detectable in the Rar1- and Sgt1-RNAi lines.
Late blight resistance evaluation
Rar1 and Sgt1 silenced plants along with several controls were evaluated for late blight resistance in environmentally controlled greenhouses at the University of Wisconsin-Madison Biotron facility. Controls include the susceptible potato cultivar Katahdin (without RB gene), S. bulbocastanum (PT29), non-transformed K41 lines and K41 lines that were transformed with the construct containing either Rar1 or Sgt1, but not silenced. The inoculation and resistance evaluation were performed as described previously [43]. Briefly, the selected lines were grown in triplicates and were randomly placed in a mist chamber eight hours prior to inoculation. The mist chamber held a 24-hour relative humidity of 100%, an 8-hour light period, a daytime temperature between 17–19°C and a nighttime temperature at 13–15°C. The plants were inoculated with 76,000–80,000 sporangia/ml of sporangial suspensions of P. infestans isolate US930287 (US-8 genotype, A-2 mating type). Measurements of the foliage blight were interpreted and scored according to the Malcolmson scale [48]. The scale was based on percent of foliage infected and scores were as follows: 9 – no visible infection; 8 – <10% infection; 7 – 11–25%; 6 – 26–40%; 5 – 41–60%; 4 – 61–70%; 3 – 71–80%; 2 – 81–90%; 1 – >90%; 0 – 100% infection. Blight scores were recorded 5, 7 and 10 days after inoculation. An average score for the resistance was determined using the three replicates of each clone in each inoculation experiment.
Resistance evaluation using a GFP-tagged P. infestans strain
A quantitative method was employed using a strain of P. infestans, which contains GFP, to better quantify pathogen growth. All silenced lines, as well as the transgenic but not silenced Rar1-RNAi and Sgt1-RNAi lines, were inoculated by the GFP-tagged P. infestans isolate 208m2 provided by Dr. Felix Mauch (University of Friberg, Switzerland) [29]. The final average sporangial concentration was 64,259 sporangia/mL, using a hemocytometer. The sporangial suspension was placed at 15°C for three hours before inoculation to release the zoospores. One 10 μl drop of the suspension was placed on both sides of the midvein, on the bottom of the leaf, in approximately the same location. Two leaves were sampled from each plant 24 and 72 hours after inoculation and four leaves were sampled 144 hours (six days) after inoculation. Each leaf was examined for the presence of actively growing Phytophthora using an Olympus SZX12 dissecting scope. Each area was photographed with an Olympus DP70 digital camera using the 41020 Chroma narrow-band GFP filter with an excitation of 425/75 nm and emission of 500/50 nm.
The spreading Phytophthora on each leaf surface were quantified by first converting the GFP images to black and white. The color conversion highlights the GFP Phytophthora sporangia and mycelium growth within each area. The area was then outlined and analyzed using the "analyze particle" tool in the ImageJ software [49]. This tool scans the image selection until it finds the edge of an object, corresponding to fluorescing sporangia or mycelium on the leaf surface. The tool outlines each object measures it and fills it in. It counts this small area as one particle and resumes scanning until it reaches another particle, where it repeats the process until the end of the image selection. The data report details the total number of counted particles, representing the total number of fluorescing sporangia on the leaf surface. As the Phytophthora grows, more sporangia and mycelium are present, which translates into a higher particle count. We measure the pathogen growth directly, not just the lesion, or area of dead tissue present on the leaf surface, which may not correspond to the actual pathogen spread. This experiment was designed as a Complete Randomized Design (CRD) with a total of 56 observations for days one and three and 50 observations for day six. Data quality tests and assessments were performed, such as residual and qq-plots. A one-way ANOVA with unequal variance was performed to compare the particle counts for each of the silenced lines with the empty vector controls and Fisher's LSD tested each group individually.
Authors' contributions
PBB and JJ conceived the project. PBB developed RNAi constructs. JAR and SA developed transgenic lines. PBB and PN characterized transgenic lines. PBB, LCK, and SMW conducted disease resistance evaluation. PBB and JJ drafted the manuscript. All authors read and approved the final manuscript.
We thank CSIRO Plant Industry(Australia) for providing the pHellsGate 8 silencing vector. This research was supported by a grant from the National Science Foundation Plant Genome Research Program (DBI-0218166) to S.A.P. and J.J. and Hatch funds to J.J.
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Huang SW, van der Vossen EAG, Kuang HH, Vleeshouwers V, Zhang NW, Borm TJA, van Eck HJ, Baker B, Jacobsen E, Visser RGF: Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato.
Plant J 2005, 42:251-261. PubMed Abstract | Publisher Full Text
Gebhardt C, Valkonen JPT: Organization of genes controlling disease resistance in the potato genome.
Annu Rev Phytopathol 2001, 39:79-102. PubMed Abstract | Publisher Full Text
Gebhardt C, Ballvora A, Walkemeier B, Oberhagemann P, Schuler K: Assessing genetic potential in germplasm collections of crop plants by marker-trait association: a case study for potatoes with quantitative variation of resistance to late blight and maturity type.
Mol Breed 2004, 13:93-102. Publisher Full Text
Park TH, Gros J, Sikkema A, Vleeshouwers V, Muskens M, Allefs S, Jacobsen E, Visser RGF, van der Vossen EAG: The late blight resistance locus Rpi-blb3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato.
MPMI 2005, 18:722-729. PubMed Abstract | Publisher Full Text
Park TH, Vleeshouwers V, Huigen DJ, van der Vossen EAG, van Eck HJ, Visser RGF: Characterization and high-resolution mapping of a late blight resistance locus similar to R2 in potato.
Theor Appl Genet 2005, 111:591-597. PubMed Abstract | Publisher Full Text
Villamon FG, Spooner DM, Orrillo M, Mihovilovich E, Perez W, Bonierbale M: Late blight resistance linkages in a novel cross of the wild potato species Solanum paucissectum (series Piurana).
Theor Appl Genet 2005, 111:1201-1214. PubMed Abstract | Publisher Full Text
Bradshaw JE, Bryan GJ, Lees AK, McLean K, Solomon-Blackburn RM: Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of Black.
Theor Appl Genet 2006, 112:744-751. PubMed Abstract | Publisher Full Text
Bradshaw JE, Hackett CA, Lowe R, McLean K, Stewart HE, Tierney I, Vilaro MDR, Bryan GJ: Detection of a quantitative trait locus for both foliage and tuber resistance to late blight [Phytophthora infestans (Mont.) de Bary] on chromosome 4 of a dihaploid potato clone (Solanum tuberosum subsp. tuberosum).
Theor Appl Genet 2006, 113:943-951. PubMed Abstract | Publisher Full Text
Rauscher GM, Smart CD, Simko I, Bonierbale M, Mayton H, Greenland A, Fry WE: Characterization and mapping of RPi-ber, a novel potato late blight resistance gene from Solanum berthaultii.
Theor Appl Genet 2006, 112:674-687. PubMed Abstract | Publisher Full Text
Simko I, Costanzo S, Ramanjulu V, Christ BJ, Haynes KG: Mapping polygenes for tuber resistance to late blight in a diploid Solanum phureja × S. stenotomum hybrid population.
Plant Breed 2006, 125:385-389. Publisher Full Text
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Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Parker JE: Regulatory role of SGT1 in early R gene-mediated plant defenses.
Science 2002, 295:2077-2080. PubMed Abstract | Publisher Full Text
Tornero P, Merritt P, Sadanandom A, Shirasu K, Innes RW, Dangl JL: RAR1 and NDR1 contribute quantitatively to disease resistance in Arabidopsis, and their relative contributions are dependent on the R gene assayed.
Plant Cell 2002, 14:1005-1015. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Si-Ammour A, Mauch-Mani B, Mauch F: Quantification of induced resistance against Phytophthora species expressing GFP as a vital marker: Beta-aminobutyric acid but not BTH protects potato and Arabidopsis from infection.
Mol Plant Pathol 2003, 4:237-248. Publisher Full Text
Muskett PR, Kahn K, Austin MJ, Moisan LJ, Sadanandom A, Shirasu K, Jones JDG, Parker JE: Arabidopsis RAR1 exerts rate-limiting control of R gene-mediated defenses against multiple pathogens.
Plant Cell 2002, 14:979-992. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pajerowska KM, Parker JE, Gebhardt C: Potato homologs of Arabidopsis thaliana genes functional in defense signaling – Identification, genetic mapping, and molecular cloning.
MPMI 2005, 18:1107-1119. PubMed Abstract | Publisher Full Text
Takahashi A, Casais C, Ichimura K, Shirasu K: HSP90 interacts with RAR1 and SGT1 and is essential for RPS2-mediated disease resistance in Arabidopsis.
Proc Natl Acad Sci USA 2003, 100:11777-11782. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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Mol Cell 1999, 4:21-33. PubMed Abstract | Publisher Full Text
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Bieri S, Mauch S, Shen QH, Peart J, Devoto A, Casais C, Ceron F, Schulze S, Steinbiss HH, Shirasu K, et al.: RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance.
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Plant Cell 2005, 17:1268-1278. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Tor M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, Mert-Turk F, Can C, Dangl JL, Holub EB: Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes.
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Helgeson JP, Pohlman JD, Austin S, Haberlach GT, Wielgus SM, Ronis D, Zambolim L, Tooley P, McGrath JM, James RV, et al.: Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight.
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Potato Res 1982, 25:213-214. Publisher Full Text

subset: commoncrawl


text:
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As our world expands to include the entire galaxy everyone rushes to claim their own piece of this new world and thousands of colonies arise. With no core government, rogue colonies begin to wreak havoc and without a centralized army to protect the innocent peace is all but lost. Now at the end of mankind s greatest battle, empires will crumble, alliances will form, enemies will rise, and heroes will fall
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OK for a view and some nice special effects but I think they were a bit short on money to have more people in it.Published on December 8, 2012 by David B Rich
maverick stinks. I could tell the first ten seconds of the film. What the !@#$% was up with thr first fight. it was so bad that i almost crapped myself laughing. Read morePublished on February 23, 2011
It's a stinker but I bought it because I was jonesing for bad Sci Fi and it most certainly was.
Plot was bad but not too terrible and acting was stiff. Read more
This is most definitely a film to be rented for a dollar instead of bought. As people have said before this is a very low-budget production. Read morePublished on August 2, 2010 by M. Glantz
The tag line promises some grand space opera story that never happens. It is NOT an exaggeration to say that a group of high school kids with a camera could come up with a better... Read morePublished on July 6, 2010 by Iron Giant
I watched the whole film. The whole thing, knowing it was obviously NOT going to get better. Sadly, it didn't really get worse. It was just "blah" bad. Read morePublished on May 17, 2010 by Samuel Mills
I'm an SF fan and am willing to tolerate some bad acting or effects if there is a story or just ANYTHING in a movie that offers some imagination. Read morePublished on May 1, 2010 by Johnny Na
From the opening credits, you know you are in for a treat.
Remember back to 7th grade when you thought how much fun it would be to make your own sci-fi movie? Read more


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I am proud of this kid. I went to the same HS(Archbishop Molloy) and I am glad that he was able to do something like this.He gave his all out on the field for his team. And that is why this team is fun to watch. Thank you Mike.
Video from the Great Catch By Mike Baxter
by Media Goon @mediagoon | Jun 2, 2012 | 0 comments


text:
CHICKASHA -- The police chief says his officer did nothing wrong when he used a taser on a man who later experience a health issue and was taken to the hospital in critical condition.
Police defend officer in tasing incident
Chickasha Police Chief Eddie Adamson told the Chickasha Express-Star it was unfortunate the man, Kevin Branson, had the medical issue and had to be hospitalized, but he said officer David Harper-Head used no improper force.
Police said Bransom, who the officer originally was trying to stop for a traffic violation, first fled from the officer by vehicle, then by foot.
The chief said Bransom then did not comply with officers and ultimately was tasered before continuing to scuffle with them.
It was when he was being handcuffed that he exhibited the medical problem.
Read the rest of this post on chickashanews.com


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|Project Name
|Price (USD)
|Project Name
|Price (USD)
|User Generated Content Website
|...functionalities to my website. It will be a user generated content website with a news feed. The basis of
|PHP, Script Install, Website Design, MySQL, HTML
|Jun 11, 2012
|Jun 11, 2012Ended
|user generated content platform
|...to users for generating/uploading and sharing content like their pics, videos or any texts with their...review or comment their content and based on the view, likes or shares their content gets , they will be
|Website Design, Social Networking
|Oct 27, 2012
|Oct 27, 2012Ended
|User generated site around sleeping
|...coded in php, with a MySQL database driving the content. Visitors should be able to create a profile...recommended #hours sleep per age (admin input) - User will see if the hours sleep per day match the recommended...of daily sleep hours based on calendar function. User can pick day to fill in the # hours sleep for that
|PHP, Project Management, Engineering, Software Architecture, Software Testing, Website Testing, MySQL, Website Management, Web Hosting
|Feb 21, 2009
|Feb 21, 2009Ended
|Wordpress User-Generated Product Comparison Site
|I need a wordpress theme to be created that will allow my users to add lists and for other users to add...and Cons of each item. So basically I want a user-generated product comparison review site. I would
|PHP, AJAX, Wordpress, CSS, HTML
|Sep 29, 2013
|Sep 29, 2013Ended
|94810 User Generated Content Site
|...that allows user to submit entries, similar to digg.com and delicious.com, but not on user submitted news...delicious.com. When submitting a entry, the user should be able to add tags, title, description,...categories in the Admin panel. For each entry, other user should be able to comment, save, email, flag it
|PHP, Website Design, AJAX, CSS, MySQL, Anything Goes
|Nov 3, 2006
|Nov 3, 2006Ended
|User generated Content Blog/Website
|CONFIDENTIAL This site is about User Generated Content! Website style examples are: http://www...allowing text comments with a user uploaded picture. Site requirements 1.User should be able to read the...the comments of previous posters 2. User must register to post comments 3. Registration fields (mandatory
|PHP, Javascript, XML, Adobe Flash, Website Design
|Jan 4, 2009
|Jan 4, 2009Ended
|Create user generated pages for my magazine
|I want to add 2 user-generated sections to my web magazine. Users will submit links with a thumbnail...delete) the user-generated content in a simple dashboard. My magazine is built on Wordpress, but I would...I need to be able to moderate the postings - The user must login (and create a username and password)
|PHP, System Admin, Project Management, Engineering, User Interface / IA, Software Architecture, Software Testing, Website Testing, MySQL, Website Management, Web Hosting
|Nov 2, 2010
|Nov 2, 2010Ended
|Member Generated Pages
|User generated content for WordPress, pages, membership, payment and user selected options. Certain options
|PHP, Wordpress
|Nov 5, 2012
|Nov 5, 2012Ended
|PHP, Website Design, Graphic Design, HTML
|Oct 29, 2012
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|Create a user generated content site
|...that associated content has. Users will be able to create a log-in, post their content, and track their...that content. I am not trying to re-invent the wheel, just trying to recreate associated content on a
|Website Design, Graphic Design, User Interface / IA
|Jun 15, 2011
|Jun 15, 2011Ended
|User generated* events listing website/community
|...using the templatic nightlife events directory theme. i would like my website to include selling of tickets...completely user friendly for myself, and registered users, so that they can produce the content and share
|PHP, Wordpress
|Jun 24, 2013
|Jun 24, 2013Ended
|User-Generated Web App
|...site will have user-generated content, and users can do various things to the content such as comment
|PHP, Python, Website Design, Graphic Design, Ruby on Rails
|Oct 1, 2008
|Oct 1, 2008Ended
|Website of user generated content and ratings
|...by other users about the content. After certain amount of good ratings the user will be rewarded with a...have no experience with Facebook Connect and custom user interfaces. BIDS will ONLY BE CONSIDERED if...requirements provide a Flow Chart of a non registered user with the following outputs: - Visiting for the
|PHP, Website Design, Graphic Design, HTML
|Jun 4, 2012
|Jun 4, 2012Ended
|Website for managing user generated texts (symfony / PHP)
|...them, read and comment them. ## Deliverables ## User accounts * Name, icon, image, description (tagable)... Privacy settings: which parts of the user's content is public, private or restricted * Create a group...it (to give them access to parts of the user's content) ## Account groups * Different types of accounts
|PHP, Javascript, Project Management, Engineering, Software Architecture, Software Testing, MySQL, Amazon Web Services
|Nov 22, 2010
|Nov 22, 2010Ended
|User-generated Google Map in Word Press
|I need a custom wordpress installation where users can place content on a Google Map. The first use... speciality and contact information. But the wordpress installation have to be made so that I can easily...a comments system. People wanting to place content on a map, has to log in using their facebook or
|Script Install, Wordpress, Twitter, Facebook Marketing
|Jul 30, 2009
|Jul 30, 2009Ended
|User generated content management website
|...Other users will be granted privileges to create content that will go into subdirectories eg. start a poll...Newly added content should be linked to the home page by order of popularity and/or when content was created...created (newest content). 4. Vendors should have the option of selecting what geographical location
|PHP, C Programming, Java, XML, Python, Script Install, Project Management, Engineering, Testing / QA, CSS, C# Programming, Software Architecture, Software Testing, Website Testing, Shell Script, MySQL, Usability Testing, Amazon Web Services, XSLT, Website Management, Web Hosting
|Oct 26, 2010
|Oct 26, 2010Ended
|Build a User Generated Content Website
|...format but will simply contain different content. Some content will be posted by the owners of the site...site, but will be built primarily through user generated content - think Trip Advisor as an example. Like
|PHP, Website Design, Graphic Design, HTML
|Aug 12, 2015
|Aug 12, 2015Ended
|Urgent: site for user-generated content
|...Quality of past work / experience building user-generated content sites. Because these are my selection
|PHP, Engineering, User Interface / IA, Software Architecture, Software Testing, MySQL
|Nov 8, 2006
|Nov 8, 2006Ended
|PHP, Website Design, Graphic Design, Wordpress
|Nov 22, 2014
|Nov 22, 2014Ended
|user generated content for travel website
|samll website like to upload and present video, for more info check attachement. ## Deliverables 1) Complete and fully-functional working program(s) in executable form as well as complete source code of all work done. 2) Deliverables must be in ready-to-run condition, as follows (depending on the nature of the deliverables): a) For web sites or other server-side deliverables intended to o...
|PHP, ASP, .NET, Windows Desktop, Engineering, Software Architecture, Software Testing, Microsoft, MySQL
|Nov 28, 2006
|Nov 28, 2006Ended


text:
Sigur Rós at Lollapalooza, 8/5/12
By Abbie Gobeli
Jack White at Lollapalooza, 8/5/12
[Slideshow] Lollapalooza 2012 Day One: The Music
[Slideshow] Lollapalooza 2012 Day Two: The Music
[Slideshow] Lollapalooza 2012 Day Three: The Music
[Slideshow] Lollapalooza 2012: The People
Sigur Rós
Lollapalooza at Grant Park, Chicago
Sunday, August 5, 2012
Sigur Rós unleashed a baptism of ethereal beauty that blanketed the tightly packed occupants of Lollapalooza's Red Bull stage on the final day of Lollapalooza. The four members recruited eight other musicians to recreate the band's heavenly sound -- including a brass section and airy background vocals.
"Svefn-g-englar," opened the performance, and slowly synched into church organ, subtle breezes of white noise, and a single repeated note that brightly rippled through the carefully layered soundscape. Frontman, Jónsi Birgisson literally licked his guitar strings -- delivering the last reverberating chords. They flawlessly transitioned into "Varúð" from their recently released sixth album,Valtari
. This lovely six-minute piece emulated enormous orchestral waves crashing with a drumming undercurrent combined with a dark, hollow synth. Resonating vocals seeped in chanting "Varúð" gently floating to the top of the surface.
Jónsi slowly motioned to the crowd to vocalize with him to "Saeglopur." As he gestured with his right hand, a row of decorated tassels dangled from the sleeve of his purple tunic. The audience quickly complied howling "oohs" in response. His face looked determined as he unveils unearthly pure falsettos.
Photo by Erik Hess
Jónsi rapidly said something in Icelandic including, "Takk fyrir" meaning "Thank you" before he and Georg exchange an eager grin while he grabs his cherry red Les Paul guitar. They continued on to "Hoppípolla," from their 2005 album, Takk..., which translates as "Jumping into puddles" in Icelandic. Though Sigur Rós combines Icelandic and Hopelandic, a language made up by the band with no semantic connotation other than it compliments their powerfully charged sound, there was no language barrier when it came to the music.
They closed with "Popplagið," a surprisingly heavy in lo-fi guitar that wails above the haphazardly falling current of instrumentation. The beaming 12 musicians bowed in curtain call fashion with Jónsi happily blowing kisses to the audience while they gasped for more.
The Crowd: Cheering, crying, and breathless with approval.
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Selected Community Events for Parades
Go Back
http://www.wtvy.com/community/events/330117311.html | 330117311
Columbia 2015 Christmas Parade
Event Dates and Times
Town of Columbia
The town of Columbia is having a Christmas Parade, with a theme honoring our veterans.
Line up starting at 5 pm on Lee Street at the parking lot next to the football field.
Acceptable unit entries are floats, cars, trucks, wagons, your feet, horses or any other modes f transportation. All bands are encouraged to attend. If you are a beauty queen (past or present or hold any kind of title we would love to have you participate.
Special invitation to church's and seniors to come and help celebrate.
Santa will be in the sunken garden after the parade. The Columbia Historical Society will be selling homemade soups and chili starting at 4 pm at the restaurant on Main Street.
For more information contact 3334.714.3033 or 334.714.9164.
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Die Zombie 4.1
In the near future, following an environmental disaster on the planet, there is a new ice age. It's the end of the world as we knew it. From the ruins of once powerful civilizations, a Corporation was founded to protect what is left of humanity. You, as a soldier of the Corporation, will carry out various missions, traveling through the ruins of the destroyed city while fighting to survive in the new, harsh world.
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Duplicate Checker 3.4
Duplicate file finder and remover. Duplicate Checker is a powerful tool for finding duplicate images, photos, mp3 and any other file types. Using this utility you can organize files on your hard disk. Main features: 1) search files with the same name, same size, same content (based on CRC32 algorithm); 2) image preview and thumbnails bar, mp3 bar; 3) many operations on list of found files: delete, move, copy; 4) export to CSV file
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Columns Master 3D 3.2
Everyone knows the Match3 game genre. Colorful crystals, berries or fruit, a fun way to pass the time. But few people know that the genre traces its root to the mid-80s, and later, in the 90s commonly found on the game consoles. Game Columns Master 3D is a remake of this game. This modern rendition of the game can be described as Tetris with match3 rules. What we have here are two completely different games mixed into one, which offers ..
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Columns Master II 4.2
Everyone knows the Match3 game genre. Colorful crystals, berries or fruit, a fun way to pass the time. But few people know that the genre traces its root to the mid-80s, and later, in the 90s commonly found on set-top boxes. Game Columns Master is a remake of this game. This modern rendition of the game can be described as Tetris with match3 rules.
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Ambers Boom 1.0
The tribe in which he lived, our hero, is located near the majestic mountains with lots of caves and grottos. One of the residents found in a cave amber stones, which immediately became appreciated above all others these ornaments. And of course many of the tribe rushed to look amber stones, including our hero. Goal of the game: Collect all amber stones, while in the caves need to dodge the enormous beetles, bats, dinosaurs and large stones.
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Forest Werewolves 2.1
One day my friend, the reporter called me and said that he was sent to the forest under the name Forest Werewolves in order to find out what is happening there. At night, he called me again and said, "They found me, these terrible, big, hungry wolves. They surrounded me. I managed to shoot them on video. Please find me in the damn woods. If I do not survive, then take away the camera and take it it to the police to these horrors ceased not to hav
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Tcca 1.6
Tcca is a very interesting game. Everyone who plays in Tcca feels the joy of a man who found a vulnerability in the opponent's position. The game "Tcca" again and again forces you to fight for victory on the nine-celled battlefield against an artificial intelligence computer. So are you ready to test your wits? Then go for it!
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Falco Sokoban 4.6
Sokoban remake. The goal is to place all the boxes into position. You will repel some kinds of monsters, which can be blown up with dynamite. Six interesting levels, each with different features. You work all night, and soon encounter monsters who want to eat you. Use the dynamite you found strategically to take care of them.
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Universe of LIFE Sound 5.1
Universe of LIFE Sound shows the 3D reproduction behavior of microorganism graphically vivid and acoustical audibly. The reproduction rules, the incremental width and the output time of the planes and generations can be free selected. Palettes can be produced on your own. With the search course, new arithmetic rules are found showing nice graphics forms.
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trakax 5.10.3
We found that being creative with video on Windows PCs was hard. Through a combination of great software, really useful resources, customer service and affordable prices, we want to allow anyone, regardless of their skill level, to create engaging original videos. Made exclusively for Windows PC.
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Find and Replace It For Windows 2.3.2
Find & Replace It! is a powerful search and replace utility. It allows performing very complex batch-replacement inside text files of any size. It supports regular expression syntax and dozens of encodings. It has scripting capabilities which allow transforming on the fly the replacement text for every found string. It even handles batch processing of the encoding of files, as well as of types of end-of-lines.
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AKick PC Optimizer 1.0.0
From Akick PC Optimizer: To stop your PC from experiencing regular crashes, freeze, and blue death screen, you need to free up your system's hard drive. Akick Optimizer is what you need for this process that allows your PC more flexibility in performing and carrying out all complicated tasks with ease. What Akick does remove all unwanted files that may be taking space on your computer's hard drive. These are mostly for useless files that you may never need at all. It also helps to organize and to rid your PC of all unnecessary files causing your PC to run too slower than a normal. All these include invalid window registry entries, shortcuts, internet stored history items, your download history, temporary files, recycle bin files and more. Smart scan report-After a whole PC check, the automatic scan software shows you each and every critical issue found and grouped into particular categories. Using the depth analysis, it also generates a quick report for all detected issues that may be some p ...
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FreeGamia Adventure Ball 1.0
In the far distant galaxy lived had the ball and called him any way. One rainy day, he wandered into the forest and got lost. In the long wanderings to find a way out of the forest he found a portal that carried him into the labyrinth from which he will get to go home. In this game you will control a ball that wants to go home. You'll have to overcome various obstacles, solve puzzles and manipulate the ball with dexterity. Each level ends with a
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Hidden Keylogger 2.19
Hidden Keylogger Pro is an advanced undetectable low level keystroke logger application that will secretly record all computer activities (visited websites, all the keystrokes, screenshots, chat convos etc.). Hidden Keylogger Pro allows you to remotely view the logs via Email and FTP. All of the computer passwords (Outlook, Internet Explorer, Firefox etc.) are successfully found and included in the logs. See more: www.hiddenkeylogger.com
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FreeGamia Star Hour HD 1.0
During high tide the Starfish found herself on the shore. Help her get to the water. You can collect the cold drops which increase maximum number of steps you can do. Whereas hot drops can decrease maximum number of steps you can do. So many different pathes but only one is correct. So many fascinating episodes with pleasant music. Starfish waiting You! It is Your Star Hour!
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Edge basis functions associated with loops forming fundamental sets on finite element facets rather than with single edges are introduced. It is shown that using these to approximate vector potentials results in less linearly interdependent equations than with usual edge basis functions. Furthermore, the conditioning of the matrix in the Galerkin equations system obtained is demonstrated to be substantially better than when using a tree-cotree gauge.
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text:
Hi to everyone,
My name is Angel.I am from Armenia. I want to share with all of you with my good experience with this agency. Firts of all I want to mention that we were looking for a good travel agency two or three months before our wedding..we made great job and eventually I read some reviews about Island Voyage Maldives here..some were reliable, good, others bad..so we were confused at first. But eventually I wrote to them.One guy Ahmed he replied and gave us very good prices for resorts...
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Thelu Veliga Retreat to open in October
Posted on Thursday August 20, 2015
Thelu Veliga Retreat will be the first offering from new Maldivian brand "Drift Retreats & Cruises". The brand seeks to bring an authentic Maldivian experience. Thelu Veliga is a tiny, 1.1 hectare, untouched gem in the South Ari Atoll offering just 30 villas. This includes 10 beach bungalows located just a few steps away from the waters. A... Read more »
The post Thelu Veliga Retreat to open in October appeared first on Maldives Travel Blog.
Inviting New Villas at Soneva Fushi
Posted on Tuesday July 28, 2015
Accommodation options available at the reputed Soneva Fushi resort in Maldives just got better with the addition of two new Villas. One of the new Villa includes three bedroom while the other Villa boasts six luxurious bedrooms. The most special feature about these two brand new Villas is the privacy on offer.... Read more »
The post Inviting New Villas at Soneva Fushi appeared first on Maldives Travel Blog.
Bollywood Diva Bipasha Basu in Maldives
Posted on Saturday July 25, 2015
Hot Bollywood Diva Bipasha Basu has visited the Maldives to take part in the Music Festival organised to promote the Visit Maldives Year 2016. She is accompanied by her lover Karan Singh Grover and friends. Bipasha Basu arrived on Saturday morning. Previously it was reported that many surprises are in store for the... Read more »
The post Bollywood Diva Bipasha Basu in Maldives appeared first on Maldives Travel Blog.


text:
Sito Web
Sei appena ritoccata uno e due camere da letto unità impostare un casual orme di sola impostazione tropicale per le belle sabbie bianche di Fort Myers Beach. Mango Street Inn offre un atmosfera calda e grazioso, la gestione in loco e incessante ospitalità. Florida certificato verde alloggio Award.
Una Carta Regalo BedandBreakfast.com è ideale per chi ha tanto bisogno di relax. Le carte sono unicamente accettate nei B&B nordamericani.
Valore della struttura124 Recensioni
Scrivi una recensione
su marzo 20, 2015
Whilewe were a bit apprehensive about staying at a B&B, when I arrived the owners Tree and Dan certainly put my wife and I at ease.. They were kind, generous and charming. The B&B was fantastic with a fresh homemade breakfast every morning. The unit was a one bedroom apartment with a full kitchen, across the stree from the beach. The B&B supplied beach chairs, towels, coolers, and umbrellas for the beach. Dan and Tree were a wealth of knowledge about activities and restaurants in the area. Most acitivites were in walking distance. We did not rent a car and decided to use the trolley which cost 75 cents one way (35 cents if you are a senior). Well worht it. We ahd to leave on an early morning flight, so Dan preoared a food package for us to take to the airport the night before. We were there for the annual shrimp festoval and St. Patrick's Day. Beleive me this place was excellent.
su novembre 18, 2014
My wife and I have now stayed at the Mango Street Inn 4x's in the last year and a half, after our first stay we decided to get married at this hidden gem. You will return from your vacation missing this joint and spending the following weeks planning your return trip.
Yes, location is great, food is wonderful, but Dan and Tree Andre make this place! Personality plus! Do not pass this place up, you will not be disappointed!
Paul and Tonya Sauer
su settembre 18, 2014
Tree and Dan provided the warmest hospitality and beautiful breakfasts. We stayed with our four year old daughter, they made us feel so welcome as a whole family with buckets and spades at the ready and really helpful advice about places to visit.
The Mango Street Inn is the best reason to visit Fort Myers Beach!
su settembre 10, 2014
I recently stayed at Mango Street Inn late August and I must say, I will definitely be back! I surprised my fiance with a trip to florida, and was so excited to find this place (from a buzz feed article!). The price and location is great, and Dan and Tree are the loveliest people.
We stayed in room 1, which was the perfect size for a couple, with a living room, small table, kitchen, bedroom, and bathroom. Everything was clean, A/C was on when we arrived, as well as cold drinks, and we had beach chairs, towels, and an umbrella all set for an early beach trip the next day. Breakfasts were fantastic, and the free bikes could have easily been mine and my fiances favorite "perk". Fort Myers itself is beautiful, the beach was right across the street and were white sand and the water was the warmest I've ever felt. We even went for a sunset dip and saw 2 dolphins swimming only a little farther out.
It is the perfect, cozy, little inn and from before even booking the room to the last night we were there, Dan and Tree provided exceptional service. Already recommended to my family and friends!
su settembre 08, 2014
We had a fantastic stay at Mango Street Inn. The property is lovely and the hosts couldn't be better. Everything was very informal and this creates a very relaxing atmosphere.
su agosto 30, 2014
This was our first stay at Mango Street, and it did not disappoint us! After spending many vacations on Sanibel we decided to branch out...boy am I glad we did! What a lovely place to spend a B'day get-away! Beautiful rooms, a short walk to the beach, wonderful hospitality from the hosts...like they are dear friends and you've know them forever...and lots of local eateries..The Beached Whale, Fresh Catch Bistro to name a few)...our next stay is booked!!
su agosto 19, 2014
We had a wonderful stay in Mango Street Inn. Dan and Tree did a fantastic job to make us feel at home. And our children loved their dog Cookie and miss it already.
The premises are nicely decorated and we appreciated the green focus of the Inn. And last but not least, breakfast. Dan and Tree did an amazing job, surprising us each morning with a tasteful meal.
We would definitely book again and longer!
su luglio 20, 2014
We had a wonderful experience here. We will be back and would recommend this bed and breakfast to anyone!
su luglio 14, 2014
We really enjoyed our stay. Tree and Dan were very helpful with anything we needed. The room was clean and it was also quiet. The beach was very close... lees than a block. Everything they advertise is true. It is a good value and a great place to stay.
su giugno 30, 2014
I just spent the most wonderful week 6/17-6/28 at the Mango Street Inn. Dan and Tree are the most friendly and charming hosts. Between Dan's gourmet breakfast and Tree's knowledge of all things in Fort Myers Beach, my week was the most easy-going vacation ever. I felt like it was my own home away from home. This place is a gem.
Vedi Altre Recensioni
Sunny and cheerful describes this one bedroom unit, thoughtfully decorated with wicker, wrought iron and glass. Soothing yellow walls surround the full kitchen, living room and queen size bedroom.
Prezzo per notte:
$105 - $165
Bright yellow walls with hints of turquoise make this room a relaxing delight. A comfy king bed and private bath complete this one bedroom unit.
This soothing and whimsical one bedroom unit has extra kitchen storage and a pull out sleep sofa in the living room.
Just like home. A large flat screen TV, comfy sofa and easy chair decorate the living area. The bedroom is finished with a wrought iron king size bed and an antique oak dresser and side table
This is a lovely bright and spacious two bedroom, one bath unit. A sunny kitchen, queen bed and two twin beds complete the space.
Prezzo per notte:
$130 - $195
This comfortable two bedroom one bath unit features a comfy leather couch, easy chairs and a full kitchen. A Queen bed in one room and two custom mahogany beds from Belize in the other.
Fronte oceano
Fort Myers, FL (RSW-Southwest Florida Regional) 15 miles
Parchi Nazionali/Statali,
Zona Naturale,
Lover's Key State Park, Matanzas Pass Preserve, Bowditch Point Park,
Punti vendita
Bed and Breakfast
BBQ , Giochi da tavolo, CD player, Computer, Fotocopiatrici, DVD Player, Scrivania, Dispositivo Fax, Telefono, VCR, Video, Attrezzature matrimoni ,
cucina elettrodomestici, mixer, processori di cibo
Aria condizionata, Sveglia , Prodotti di cortesia, Ventilatori, Attrezzature per Caffè/Tè, Frigorifero, Asciugacapelli, Accesso a internet , Biblioteche, Microonde, Ingresso privato, Radio, TV via cavo/satellite, TV, Wi-Fi, Internet via cavo
Bici, Osservazione Uccelli, Nautica, Canottaggio, Pesca, Golf, Kayak, Regate, Shopping, Scenari, Nuoto, Sport acquatici
Dan and Tree Andre
Dan è uno chef e Tree è un sommelier. Questa passione per il cibo e il vino è in primo piano del Mango Street Inn con cucina socials vino occasionale, degustazioni di vino e classi
Plus continentale
A basso contenuto di grassi , Restrictive , Vegetariano
$95 a notte
Proprietari: Dan and Tree Andre
Ospiti: 2
Cos'è il Miglior Prezzo -- Garanzia di Nessun Costo?Il Miglior Prezzo -- Garanzia di Nessun Costo copre qualsiasi prenotazione online Bed and Breakfast attraverso BedandBreakfast.com. Il Bed and Breakfast deve essere prenotato usando il pulsante per qualificarsi.
Come traggo vantaggio dal Miglior Prezzo -- Garanzia di Nessun Costo?Chiama il numero 1-844-271-6829 entro 24 ore dalla prenotazione e comunica al rappresentante dell'assistenza clienti i dettagli del prezzo più basso che trovate.
Termini e Condizioni del Miglior Prezzo -- Garanzia di Nessun CostoIl Miglior Prezzo -- Garanzia di Nessun Costo di BedandBreakfast.com assicura che quando prenoti e paghi la tua prenotazione online tramite il sito web BedandBreakfast.com, stai prenotando la tua camera al prezzo più basso possibile. Se trovi un prezzo più basso per la stessa camera, le stesse date e presso lo stesso B&B contattaci entro 24 ore dalla tua prenotazione e ti rimborseremo la differenza. Tale garanzia non si applica a prenotazioni effettuate per le festività e/o eventi particolari. Tutte le richieste sono soggette a verifica da parte di BedandBreakfast.com. BedandBreakfast.com non accetterà schermate o altra prova presunta di un prezzo più basso che non può essere confermato indipendentemente dal personale di BedandBreakfast.com. Né BedandBreakfast verificherà qualsiasi richiesta che ritenga, a sua sola discrezione, sia il risultato di una stampa o di altro errore, o di azione fraudolenta o fatta in cattiva fede.


text:
The month of June seemed not very prolific, as 6 persons with disabilities were employed in various areas of work. 3 months back we introduced a recruitment fee to companies that employ/use our services for recruitment; so far we have had a few companies agree to pay, but have not had their staff commence work. The NOWPDP team is concerned that companies are not willing to pay for the service, and therefore delaying the commencement of candidates. We will review the progress in the coming months.
Our target of companies to be met has also been increased.
A total number of 4 new companies were met:
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Executive Officer


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Responsibilities include but are not limited to: arranges for the efficient and orderly pre-admission and/or admission of InPatients and OutPatients. Insures that accurate patient information is collected and that patients are aware of hospital policies and procedures. Must be able to perform jobs in all patient care and operational areas subject to reasonable accommodations. This is a union position.
Education: High school graduate or equivalent preferred. Completion of Office Skill educational program preferred.
Experience: 5 years experience in a clerical hospital setting preferred. Good oral and written communication skills. Accurately typing at least 35 words per minute preferred. Minimum of one (1) year computer knowledge. Medical terminology preferred.
Licenses/Certificates: None


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Istanbul defends 2020 bid strength
Updated: June 12, 2013, 2:09 PM ETAssociated Press
ISTANBUL -- The anti-government protests in Turkey "will have no bearing" on Istanbul's ability to host the 2020 Olympics, the bid committee said Wednesday.
The Olympic bid has been rattled by the nearly two weeks of protests that began in Istanbul and spread to dozens of other cities. It's the biggest test yet in the 10-year rule of Prime Minister Recep Tayyip Erdogan.
Istanbul, making its fifth bid for the Olympics, is competing against Madrid and Tokyo. The IOC will select the ...
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Starship Size Argument thread
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May 3 2013, 09:28 PM
Rear Admiral
Location: In the bleachers
Re: Starship Size Argument thread
How about this: instead of using rulers and pictures and approximate scales, just Google "size of the enterprise 2009" or something like that and you'll find several links that discuss the scaling up of the ship from its length of about 1200 feet early in production to what its length became (2397 feet). There's no confusion about it. The story is widely known. Making a case for the ship being smaller is barking up the wrong tree, beating a dead horse, getting the wrong end of the stick, and all that.
Whenever you find yourself on the side of the majority, it is time to pause and reflect. -- Mark Twain
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Additional file 3.
Figure S2. iHS values in the region including each gene of the Asparagine N-Glycosylation pathway. For each gene, the iHS of all the SNPs within 400 kb upstream and 400 kb downstream are shown, on a row for each population. The values for SNPs within the gene are shown in darker grey. A smoothing line (calculated with the Loess function) is shown to help the visualization.
Format: PDF Size: 905KB Download file
This file can be viewed with: Adobe Acrobat Reader
Dall'Olio et al. BMC Evolutionary Biology 2012 12:98 doi:10.1186/1471-2148-12-98


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Debenhams Hollowfibre luxury quilted mattress protector
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Backyard Orchard News
This Bug's for You at the Bohart
If you're into bugs, the Bohart Museum of Entomology on the UC Davis campus has plenty of...
Toby and a Walking Stick
Speedy Crawler
On the Trail of Dengue
Medical entomologist Thomas Scott (right), professor of entomology at the University of California,...
Killer Mosquito
At work
What Entomologists Do to Have Fun
Ghouls just like to have fun at Halloween. So do entomologists. When the Bohart Museum of...
Matan Shelomi
Bob Kimsey, Where Are You?
Queen Bee
Citrus leafminer is everywhere!
In the San Joaquin Valley, the warm weather has stimulated quite a bit of fall flush. Citrus leafminer loves these new leaves and is attacking all varieities of citrus vigorously. While the damage is unsightly, most varieties of mature trees can take this damage and continue to grow at a normal rate. Nursery trees, new plantings, limes, and lemons with multiple crops in coastal California need insecticides to reduce the damage.
A Fly By
What's a fly doing there?Just soaking up the sun.A fly that landed on one of the two colorfully...
Between the Branches
Fly By